Reagent mixture and composition for treating red blood cells to effect sphering

ABSTRACT

A composition is disclosed of blood serum sample preparation for improved, more accurate and precise, electrooptical method for measuring erythrocyte volumes, individually and as an average.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 425,506, nowabandoned, which was a continuation of application Ser. No. 158,124, nowabandoned, which was a continuation of application Ser. No. 802,956, nowabandoned, which was a continuation of application Ser. No. 510,824,which was a division of application Ser. No. 277,539, now U.S. Pat. No.4,412,004.

BACKGROUND OF THE INVENTION

This invention relates generally to a method for sphering or spheringand fixing whole blood erythrocytes without volume change for accurateand precise cell volume measurement. More particularly, the methodemploys a series of dilution steps whereby a protein, externallyprovided or endogenously supplied and a sphering agent are added in aprotein/sphering agent weight ratio of from about 20:1 to about 70:1,based on total sample volume, and the concentration of detergent in thefinal sample is from about 2 mg./100 ml. to about 10 mg./100 ml.

Methods which utilize the measured amount of light scattered fromindividual red cells (erythrocytes) to determine the individual and meanvolumes of red cells, suffer from two kinds of errors:

1. The native human red cell is a biconcave disc and the amount of lightscattered within a particular solid angle varies with the orientation ofthe cell with respect to the incident light beam.

2. During handling, i.e. dilution and pumping, the shape of the cellscan change depending in part on the time between the drawing of theblood and the time of measurement and in part on the composition of thediluted blood sample.

For a discussion of the above, see Hemolysis and Related Phenomena,Chapter II, pp 10-49 by Eric Ponder (1948) and Transformation andRestoration of Biconcave Shape of Human Erythrocytes Induced byAmphiphilic Agents and Changes of Ionic Environment, Biochemica Et.Biophy. Acta, Bernard Deuticke, pp 494-500 (1968).

SUMMARY OF THE INVENTION

The present invention eliminates both of these sources of error andpermits vastly improved methods for determination of human red bloodcell volumes. It is well known, see for example Ponder supra, that it ispossible to sphere red blood cells in isotonic solution without changingtheir volumes. Since the light scattering from a perfectly sphered cellis invariant with orientation in a light beam, the first kind of erroris eliminated. However, such preparations are notoriously unstable andred cell lysis occurs at various times after sphering, depending on thechoice of sphering agent and the properties of the individual bloodsamples.

It has now been discovered that prolonged stability of the sphered statecan be achieved by controlling the absolute concentration of thesphering agent (typically a material with detergent properties) and theweight ratio of sphering agent to protein, either added or endogenous atany desired dilution in isotonic solution. This helps to assure shapeconsistency during processing and minimizes the second kind of error.

The method of this invention can be carried out generally in two ways:

A. A blood sample is diluted, typically about 1/1000, in an isotonicsolution containing sphering agent (detergent) and albumin at therequired concentrations; or

B. The blood sample is diluted with an amount of isotonic solutioncontaining the sphering agent at a concentration which is justsufficient to cause sphering when the dilution provides the correctratio of sphering agent to the endogenous serum albumin (plasma protein)from the blood sample itself. The resulting sample is thensimultaneously and/or successively fixed and further diluted by addingan isotonic solution of a fixing agent to harden the sphered cells andmake them completely insensitive to processes which could otherwisecause them to change their shape or size or lyse and lose theircontained hemoglobin.

In accordance with this invention, there is claimed a method fortreating mammalian red blood cells in a sample to provide a sample whichcan be effectively measured electrooptically for determination of redblood cell volumes which comprises combining an anticoagulated wholeblood sample with an isotonic solution containing sphering agent, anddiluting an aliquot of the resulting sample with an isotonic solutioncontaining protein and sphering agent. The weight ratio in the finalsample of protein/sphering agent is from about 20:1 to about 70:1,preferably about 50:1 and the concentration of sphering agent is fromabout 2 mg./100 ml. to about 10 mg./100 ml., preferably about 3 mg./100ml.

Preferably, whole blood sample is prediluted with saline, as diluent,resulting in about a 50% by volume dilution of sample to reduceviscosity and therefore assure reduction of volumetric pumping errorswhich stem from variations in blood sample viscosities. The subsequentdilution steps result in a final dilution of sample of about 1:1000 byvolume to produce a dilution such that the probability of more than onecell passing through the incident light beam of the electroopticaldetector during the detector's measuring time window is very low.

The detergent used in this method is preferably an alkali metal salt ofan alkyl sulfate, said alkyl group containing from 10 to 16 carbonatoms. Sodium lauryl sulfate is most preferred.

The protein used in this method is preferably serum albumin, which isadded externally.

Another preferred method of this invention is similar to theabove-described method except that in lieu of the protein/sphering agentdilution step, the aliquot sample is treated with a fixing agentsolution, preferably an isotonic glutaraldehyde-containing salinesolution. In this method, the protein required is endogenously providedin the sample as plasma protein.

In another preferred embodiment of this invention, there is claimed areagent for sphering red blood cells in a sample comprising aprotein-sphering agent mixture whereby the weight ratio of protein tosphering agent is from about 20:1 to about 70:1 and the totalconcentration of sphering agent in the composite sample is from about 2mg./100 ml. to about 10 mg./100 ml.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of sphering mammalian redblood cells in an anticoagulated whole blood sample. The method involvesthe employment of a protein and a sphering agent in a specified weightratio and a certain final sphering agent concentration.

In the absence of protein, after addition of a sphering agent, theamount of free sphering agent in solution is dependent upon theconcentration of red cells (see Ponder above). Therefore, with a reagentwith fixed optimal sphering agent concentration for a normal bloodcount, the degree of sphering could be either incomplete, with a bloodwith high red cell count per unit volume of solution, or could lead tolysis with a very low red blood cell count. Proteins, such as serumalbumins, bind sphering agent reversibly and can therefore be used tobuffer the effective concentration of sphering agent in the optimalrange, independent of red cell count.

The preferred concentration of sphering agent is that amount which isjust sufficient to cause sphering when buffered with a protein such asalbumin or plasma protein at any particular dilution of sample. Theprotein albumin can be provided in either of two ways: by outsideaddition or endogenously as plasma protein in the serum sample.

In a preferred embodiment of this invention, the method involvescombining a prediluted blood sample with an isotonic spheringagent-saline solution and then treating an aliquot thereof with aprotein-sphering agent saline solution.

Preferably, the predilution step is carried out by diluting the serumsample about 50% by volume with a suitable isotonic diluent such as asaline solution. The resulting prediluted sample is combined with anisotonic solution containing a sphering agent (sometimes referred toherein as detergent). A typical first dilution results in a 50:1dilution of sample. A further dilution is effected by treating analiquot of the above sample with a protein-sphering agent solution toprovide a dilution of sample of about 1000:1. The resulting samplecontains sphered and stabilized erythrocytes at a feasible concentrationfor light scattering measurement. When such light scattering measurementis conducted employing a flow cell cytometer, the individual cellvolumes can be determined as well as the number of cells. The meanvolume can therefore also be calculated.

A critical feature of this method involves the weight ratio ofprotein/sphering agent and the concentration of sphering agent. Byregulating these parameters within certain limits, the sphering processis effectively accomplished and the analytical results highly probative.

It has been found that a weight ratio of protein/sphering agent in theherein disclosed method is preferably from about 20:1 to about 70:1,with a ratio of 50:1 most preferred. For the final concentration ofsphering agent, a concentration of from about 2 mg./100 ml. to about 10mg./100 ml. is highly suitable, with a concentration of 3 mg./100 ml.most preferred.

The protein, externally supplied, is preferably a serum albumin. Otheremployable proteins include bovine, human and egg albumin.

In a second method of this invention, the protein/sphering agent seconddilution step is replaced by treatment with an isotonic fixing agentsolution. In this system, the protein for the first dilution is providedin endogenous form in the serum sample as plasma protein. An isotonicsolution of a sphering agent is added in a volume sufficient to bringthe endogenous plasma protein/sphering agent ratio and also theconcentration of sphering agent within the preferred ranges. Thepreferred fixing agent is glutaraldehyde, used in an amount to provide afinal glutaraldehyde concentration of from 0.1% to 0.4% by weight. Theisotonic fixing agent solution is suitably formulated with saline or asaline-sphering agent mixture.

Because glutaraldehyde fixes red cells very rapidly, optimal bufferingof the sphering agent concentration beyond the fixing agent additionstep is considered less critical. As soon as the red cell content hasbeen fixed, it becomes completely noncritical.

The sphering agent employed in either method is suitably an alkali metal(sodium, potassium, lithium, cesium or rubidium) salt of an alkylsulfate wherein said alkyl contains from 10 to 16 carbons. Alkali metallauryl sulfates are preferred, and sodium lauryl sulfate most preferred.Other suitable sphering agents which may be employed in these methodsinclude fatty acids, phospholipids, etc. It is to be noted that somenominal "sphering agents" such as crude egg lecithin (see Ponder above)actually contain a sphering agent as a minor impurity. For example, purelecithin is not a sphering agent. It is to be understood that the weightconcentrations discussed are of the active principle in any impure"sphering agent" and not the crude weight concentration.

Both methods can be effected either continuously as in an automatedsystem or in a discontinuous or discrete manner.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 represents a schematic flow sheet of the continuous system orapparatus according to one embodiment of the present invention for thetreatment of a serum sample for eventual electrooptical measurement.

DETAILED DESCRIPTION OF THE DRAWING

Referring to the sole figure, a system is illustrated for measuring thevolume of individual red cells in a discrete anticoagulated blood sampletreated in accordance with the present invention. However, it is withinthe contemplation of the present invention that the measurement of thevolume of red cells in successive anticoagulated blood samples may beeffected on a continuous basis, for example, as described in U.S. Pat.No. 3,740,143, assigned to a common assignee.

Such system comprises a peristaltic pump 1 including pump tubes 3, 5, 7,9 and 10. As is understood, the relationship of the internal diametersof such pump tubes determines the proportioning of the sample andreactants introduced into the system. An aspirating probe 13 isconnected along conduit 14, to the inlet of pump tube 5, whose outlet isconnected to a junction 15. Probe 13 is adapted to be immersed into ananticoagulated blood sample 17 contained in a sample receptacle 19. Itwill be appreciated that probe 13 may be adapted, as described in U.S.Pat. No. 3,740,143, to be immersed, in turn, into successive samplereceptacles, so as to effect the measurement of the red cell volumes ofsuccessive samples on a continuous basis.

Also, the inlet end of pump tube 3 is connected to a source 21 ofappropriate diluent for effecting the first dilution of the sample 17.Upon operation of pump 1, diluent is passed along pump tube 3 tojunction 23 in conduit 14, so as to be mixed with and dilute the samplebeing passed from the probe 13. Also, an air line 25 from an "air-bar"structure 26, as described in U.S. Pat. No. 3,306,229, assigned to acommon assignee, whose operation is phased to that of the pump 1 asindicated by the dashed connector, operates periodically to introduceoccluding air segments into conduit 14. The presence of such"intra-sample" air segments insures proper proportioning of the sampleand reactants into the system (and effective wash between successivesamples) as described in the referenced patent. Concurrently, anisotonic solution containing the sphering agent is passed from source 27along pump tube 7 to junction 15, whereat it is mixed with the dilutedsample passed along pump tube 5, to effect the second dilution of sample17. The sample is flowed from junction 15 and through mixing coil 29, toeffect a thorough mixing thereof, and subsequently along conduit 31 to aresampling fitting 33. Fitting 33 includes a waste outlet 35 and aresampling outlet 37 connected to the inlet of pump tube 9. The samplepasses from outlet 37 and to junction 39 along pump tube 9, excesssample and "intra-sample" air segments introduced into fitting 33 beingpassed to waste along waste outlet 35. A second "air-bar" structure 38reintroduces "intrasample" air segments along air line 36 into thediluted sample stream.

The inlet of pump tube 10 is connected to a source 41 of fixing agent.The outlet of pump tube 10 is connected to junction 39, whereat thefixing agent and the twice-diluted sample are mixed and passed to mixingcoil 43, to insure mixing of the same. The outlet of mixed coil 43 ispassed to a resampling fitting 45, which includes a waste outlet 47 anda resampling outlet 49, the latter being connected to the inlet of thesingle pump tube of a secondary peristaltic pump 51. The sample ispassed from the outlet 49 and through pump 51 to a sheath-streamparticle counter 53, of the type described in U.S. Pat. No. 3,740,143,supra. Again, excess sample and the "intra-sample" air segments arepassed to waste along waste outlet 47. In counter 45, the red cells inthe treated blood sample are confined to flow serially, so as to beindividually counted and their volumes measured. The treated bloodsample is thereafter passed to waste. The sphering of the red cells,according to the present invention, insures that the measured volume isindependent of the orientation of the red cells as they progress throughcounter 53. In prior art, where the red cells were not properly sphered,the random orientation of the red cells proceeding through the particlecounter often resulted in inaccurate volume determinations.

EXAMPLE I

A sample (0.37 ml.) of anticoagulated whole blood is prediluted withisotonic saline (0.23 ml.). An aliquot (0.16 ml.) of the resultingsample is combined with 4.2 ml. of an isotonic saline solutioncontaining sodium lauryl sulfate (3 mg./100 ml.). An aliquot (0.16 ml.)of the resulting diluted sample is then treated with 4.0 ml. of anisotonic saline solution containing bovine serum albumin (0.1%) andsodium lauryl sulfate (3 mg./100 ml.). The final sample is placed in aflow cell and electrooptically measured. The red blood cell count andred blood cell volume were recorded.

EXAMPLE II

A sample (0.37 ml.) of anticoagulated whole blood is prediluted withisotonic saline (0.23 ml.). An aliquot (0.16 ml.) of the resultingsample is combined with 4.2 ml. of an isotonic saline solutioncontaining sodium lauryl sulfate (3 mg./100 ml.). An aliquot (0.16 ml.)of the resulting diluted sample is then treated with 4.0 ml. of anisotonic saline solution containing glutaraldehyde (0.2%) and sodiumlauryl sulfate (1 mg./100 ml.). The final sample is placed in a flowcell and electrooptically measured. The red blood cell count and redblood cell volume were recorded.

It should be understood by those skilled in the art that variousmodifications may be made in the present invention without departingfrom the spirit and scope thereof as described in the specification anddefined in the appended claims.

What is claimed is:
 1. A composition for use in a cytometer,comprising:(a) an anticoagulated whole blood sample aliquot, and (b) areagent mixture consisting essentially of:(i) an isotonic aqueoussolution; (ii) a sphering agent; and (iii) a protein, which reversiblybinds said sphering agent;said protein and sphering agent being presentin a weight ratio of protein to sphering agent from about 20:1 to about70:1, and said sphering agent having a final concentration in the rangeof about 2 mg per 100 ml to about 10 mg per 100 ml in said reagentmixture.
 2. The composition of claim 1, wherein said protein is a serumalbumin.
 3. The composition of claim 74, wherein said detergent is analkali metal salt of an alkyl sulfate.
 4. The composition of claim 3,wherein said alkali metal salt is an alkali metal lauryl sulfate.
 5. Thecomposition of claim 4, wherein said alkali metal laurel sulfate issodium lauryl sulfate.
 6. The composition of claim 74, wherein saiddetergent is an alkali metal salt of an alkyl sulfate, said alkylcontaining from 10 to 16 carbon atoms.
 7. The composition of claim 6,wherein said alkyl sulfate salt is an alkali metal lauryl sulfate. 8.The composition of claim 7, wherein said alkali metal lauryl sulfate issodium lauryl sulfate.
 9. The composition of claim 1, wherein theconcentration of sphering agent is about 3 mg per 100 ml.
 10. Thecomposition of claim 1, wherein said protein and sphering agent arepresent in a weight ratio of protein to sphering agent of about 50:1.11. The composition of claim 1, wherein said protein is selected fromthe group consisting of bovine serum albumin, human albumin, and eggalbumin.
 12. The composition of claim 1, wherein said protein isendogenous in a whole blood sample.
 13. The composition of claim 1,wherein said sphering agent is a detergent.
 14. The composition of claim1, wherein said sphering agent is a phospholipid.
 15. The composition ofclaim 1, wherein said sphering agent is a fatty acid.
 16. Thecomposition of claims 1, 13, 14, or 15, wherein said reagent mixturefurther includes a fixing agent.
 17. The composition of claim 16,wherein said fixing agent is gluteraldehyde present in said reagentmixture in an amount providing a final gluteraldehyde concentration offrom about 0.1% to 0.4% by weight.
 18. A reagent mixture for spheringred blood cells in an anticoagulated whole blood sample, consistingessentially of:(i) an isotonic aqueous solution; (ii) a sphering agent;(iii) a protein, which reversibly binds said sphering agent;said proteinand sphering agent being present in said mixture in a weight ratio ofprotein to sphering agent from about 20:1 to about 70:1, and saidsphering agent having a final concentration in the range of about 2 mgper 100 ml to about 10 mg per 100 ml in said reagent mixture.
 19. Thereagent mixture of claim 18, wherein said protein is a serum albumin.20. The reagent mixture of claim 18, wherein said sphering agent is analkali metal salt of an alkyl sulfate.
 21. The reagent mixture of claim20, wherein said alkyl sulfate salt is an alkali metal lauryl sulfate.22. The reagent mixture of claim 21, wherein said alkali metal laurylsulfate is sodium lauryl sulfate.
 23. The regent mixture of claim 18,wherein said sphering agent is an alkali metal salt of an alkyl sulfate,said alkyl sulfate containing from 10 to 16 carbon atoms.
 24. Thereagent mixture of claim 23, wherein said alkyl sulfate salt is analkali metal lauryl sulfate.
 25. The reagent mixture of claim 24,wherein said alkali metal lauryl sulfate is sodium lauryl sulfate. 26.The reagent mixture of claim 18, wherein the concentration of spheringagent is about 3 mg per 100 ml.
 27. The reagent mixture of claim 18,wherein said protein and sphering agent are present in a weight ratio ofprotein to sphering agent of about 50:1.
 28. The reagent mixture ofclaim 18, wherein said protein is selected from the group consisting ofbovine serum albumin, human albumin, and egg albumin.
 29. The reagentmixture of claim 18 wherein said protein is endogenous in a whole bloodsample.
 30. The reagent mixture of claim 18, wherein said sphering agentis a detergent.
 31. The reagent mixture of claim 18, wherein saidsphering agent is a phospholipid.
 32. The reagent mixture of claim 18,wherein said sphering agent is a fatty acid.
 33. The reagent mixture ofclaims 18, 30, 31, or 32, further including a fixing agent.
 34. Thereagent mixture of claim 33, wherein said fixing agent is gluteraldehydepresent in an amount providing a final gluteraldehyde concentration offrom about 0.1% to 0.4% by weight.